RESUMEN
Lactam cross-links have been employed to stabilize the helical secondary structure and enhance the activity and physiological stability of antimicrobial peptides; however, stabilization of ß-sheets via lactamization has not been observed. In the present study, lactams between the side chains of C- and N-terminal residues have been used to stabilize the ß-sheet conformation in a short ten-residue analogue of chicken angiogenin-4. Designed using a combination of molecular dynamics simulations and Markov state models, the lactam cross-linked peptides are shown to adopt stabilized ß-sheet conformations consistent with simulated structures. Replacement of the peptide side-chain Cys-Cys disulfide by a lactam cross-link enhanced the broad-spectrum antibacterial activity compared to the parent peptide and exhibited greater propensity to induce proinflammatory activity in macrophages. The combination of molecular simulations and conformational and biological analyses of the synthetic peptides provides a useful paradigm for the rational design of therapeutically active peptides with constrained ß-sheet structures.
Asunto(s)
Antibacterianos , Simulación de Dinámica Molecular , Animales , Antibacterianos/farmacología , Antibacterianos/química , Antibacterianos/síntesis química , Péptidos Antimicrobianos/química , Péptidos Antimicrobianos/farmacología , Péptidos Antimicrobianos/síntesis química , Estructura Secundaria de Proteína , Pruebas de Sensibilidad Microbiana , Ratones , Lactamas/química , Lactamas/farmacología , Lactamas/síntesis química , Pollos , Péptidos Cíclicos/química , Péptidos Cíclicos/farmacología , Péptidos Cíclicos/síntesis química , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/farmacología , Reactivos de Enlaces Cruzados/químicaRESUMEN
Aberrant aggregation of proteins into poorly soluble, toxic structures that accumulate intracellularly or extracellularly leads to a range of disease states including Alzheimer's, Parkinson's, Huntington's, prion diseases, and type II diabetes. Many of the disease-associated amyloidogenic proteins are intrinsically disordered, which makes their experimental investigation challenging due to a limited number of experimental observables to effectively characterize their ensemble of conformations. Molecular dynamics simulations provide dynamic information with atomistic detail, and are increasingly employed to study aggregation processes, offering valuable structural and mechanistic insights. In this chapter, we demonstrate the use of all-atom molecular dynamics simulations to model the self-aggregation of a six-residue amyloidogenic peptide derived from amyloid ß, a 39-43 residue-long peptide associated with the pathogenesis of Alzheimer's disease. We provide detailed instructions on how to obtain the initial monomer conformations and build the multichain systems, how to carry out the simulations, and how to analyze the simulation trajectories to investigate the peptide self-aggregation process.
Asunto(s)
Proteínas/metabolismo , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Humanos , Enfermedad de Huntington/metabolismo , Simulación de Dinámica Molecular , Agregado de Proteínas/fisiología , Conformación Proteica , SolubilidadRESUMEN
Hyper-activated STAT5B variants are high value oncology targets for pharmacologic intervention. STAT5BN642H, a frequently-occurring oncogenic driver mutation, promotes aggressive T-cell leukemia/lymphoma in patient carriers, although the molecular origins remain unclear. Herein, we emphasize the aggressive nature of STAT5BN642H in driving T-cell neoplasia upon hematopoietic expression in transgenic mice, revealing evidence of multiple T-cell subset organ infiltration. Notably, we demonstrate STAT5BN642H-driven transformation of γδ T-cells in in vivo syngeneic transplant models, comparable to STAT5BN642H patient γδ T-cell entities. Importantly, we present human STAT5B and STAT5BN642H crystal structures, which propose alternative mutation-mediated SH2 domain conformations. Our biophysical data suggests STAT5BN642H can adopt a hyper-activated and hyper-inactivated state with resistance to dephosphorylation. MD simulations support sustained interchain cross-domain interactions in STAT5BN642H, conferring kinetic stability to the mutant anti-parallel dimer. This study provides a molecular explanation for the STAT5BN642H activating potential, and insights into pre-clinical models for targeted intervention of hyper-activated STAT5B.
Asunto(s)
Linfocitos Intraepiteliales , Leucemia de Células T/genética , Linfoma de Células T/genética , Mutación , Factor de Transcripción STAT5/genética , Animales , Neoplasias Hematológicas/genética , Humanos , Ratones , Ratones Transgénicos , Simulación del Acoplamiento Molecular , Dominios Homologos srcRESUMEN
Cancer-associated thrombosis is managed by the administration of anticoagulants and antithrombotic agents that have a high risk of inducing hemorrhagic complications. To develop safer strategies for antithrombotic therapy, in vivo activators of the intrinsic pathway, namely, cell-free nucleic acids (DNA and RNA) have been targeted with cationic, polyamine-based polymers. The cytotoxicity of the highly cationic polymers is a major drawback for their practical use, and biocompatible alternatives are in high demand. In this study, we carried out all-atom molecular dynamics simulations to systematically examine the DNA binding of polyamine-poly(ethylene glycol) (PEG) diblock polymers designed from biocompatible building blocks to inhibit the procoagulant activity of DNA. The differences in cationic charge, PEG chain length, and initial conformations of the polymers resulted in marked differences in their binding to DNA. We found that having an exposed cationic polyamine group is essential to polymer-DNA binding and a certain level of electrostatic interactions is necessary to maintain the bound state. Intrachain associations between the polyamine groups and PEG chains in some cases have led to a collapsed state of the polymer that precludes binding to DNA. This self-association is mainly due to a strong hydrogen bond between polymer polyamine and PEG groups and partly due to a partially charged semibranched polyamine group architecture. As polymer "masking" of DNA is thought to arrest DNA's prothrombotic activity, our findings highlight the desired structural features of the polymers for stronger DNA binding and provide insights into the design of novel antithrombotic agents.
Asunto(s)
Simulación de Dinámica Molecular , Cationes , Humanos , Ácidos Nucleicos , Polietilenglicoles , Polímeros , TrombosisRESUMEN
Delivery of polynucleotide-based therapeutics into target cells involves interactions with glycosaminoglycan chains that are located on cell membrane milieu. Mechanisms governing glycosaminoglycan-mediated changes in the nanoparticulate structures of polymer-polynucleotide complexes are unknown, and cannot be fully elucidated without atomistic level details of molecular interactions. We selected a representative nanoparticulate system consisting of a short interfering RNA (siRNA)-polyethylenimine complex, and performed all-atom molecular dynamics simulations with the prototypical glycosaminoglycan heparin. We monitored the binding between the complex constituents and the heparin, and identified key features contributing to the response of the siRNA nanoparticles to heparin. We observed three main metastable states that the siRNA nanoparticles might visit in the presence of heparin, which can be translated into different functional outcomes. By correlating our data with the widely different and seemingly contradictory roles previously assigned to glycosaminoglycans, this study provides unique insights into the discrepancies in the experimental literature concerning the role of glycosaminoglycans in the polymeric nanoparticle delivery.
Asunto(s)
Glicosaminoglicanos/química , Simulación de Dinámica Molecular , Nanopartículas/química , Ácidos Nucleicos/química , Polímeros/química , Heparina/química , Conformación Molecular , Polietileneimina/química , ARN Interferente PequeñoRESUMEN
Leukemias arise from genetic alterations in normal hematopoietic stem or progenitor cells, leading to abnormal blood population with transformed cells. With the advent of RNAi and its pharmacological mediator siRNA, it has become possible to downregulate specific drivers causing leukemias. In this review, we present unique aspects of RNAi-mediated therapy and delivery technologies. Recent updates on molecular targets and delivery systems are discussed emanating from in vitro cell models and preclinical animal models. We conclude with a view on the future of RNAi in leukemia therapy, emphasizing possible measures to achieve higher efficacy and improved safety.
Asunto(s)
Leucemia/terapia , Interferencia de ARN , ARN Interferente Pequeño/administración & dosificación , Animales , Terapia Genética , Humanos , Leucemia/genéticaRESUMEN
High molecular weight (HMW) polyethylenimine (PEI) is one of the most versatile nonviral gene vectors that was extensively investigated over the past two decades. The cytotoxic profile of HMW PEI, however, encouraged a search for safer alternatives. Because of lack of cytotoxicity of low molecular weight (LMW) PEI, enhancing its performance via hydrophobic modifications has been pursued to this end. Since the performance of modified PEIs depends on the nature and extent of substituents, we systematically investigated the effect of hydrophobic modification of LMW (1.2 kDa) PEI with a short propionic acid (PrA). Moderate enhancements in PEI hydrophobicity resulted in enhanced cellular uptake of polyplexes and siRNA-induced silencing efficacy, whereas further increase in PrA substitution abolished the uptake as well as the silencing. We performed all-atom molecular dynamics simulations to elucidate the mechanistic details behind these observations. A new assembly mechanism was observed by the presence of hydrophobic PrA moieties, where PrA migrated to core of the polyplex. This phenomenon caused higher surface hydrophobicity and surface charge density at low substitutions, and it caused deleterious effects on surface hydrophobicity and cationic charge at higher substitutions. It is evident that an optimal balance of hydrophobicity/hydrophilicity is needed to achieve the desired polyplex properties for an efficient siRNA delivery, and our mechanistic findings should provide valuable insights for the design of improved substituents on nonviral carriers.
Asunto(s)
Vectores Genéticos , Ácidos Nucleicos/química , Polietileneimina/química , Carbono/química , Simulación por Computador , Silenciador del Gen , Técnicas de Transferencia de Gen , Proteínas Fluorescentes Verdes/química , Humanos , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Células K562 , Simulación de Dinámica Molecular , Peso Molecular , Propionatos/química , Ingeniería de Proteínas/métodos , ARN Interferente Pequeño/metabolismo , Propiedades de SuperficieRESUMEN
Delivery of small interfering RNA (siRNA) for silencing of aberrantly expressed genes is a promising therapy for the treatment of various genetic disorders. Polymeric carriers have been used in the design of efficient delivery systems to generate nanoscale siRNA polyplexes. Despite the great amount of research pursued on siRNA therapeutics, the underlying mechanisms of polyplex dissociation in cytosol are still unclear. The fate of siRNA polyplexes during intracellular stages of delivery and how the endogenous molecules may affect the integrity of polyplexes remains to be explored. In this study, we have focused on miRNA-21 as a representative anionic endogenous molecule and performed gel electrophoresis mobility shift assays, particle size and zeta (ζ)-potential analyses, and a series of all-atom molecular dynamics simulations to elucidate the effect of miRNA on siRNA-PEI polyplexes. We report a slightly better binding to PEI by miRNA than that of siRNA, and speculated that miRNA may disrupt the integrity of preformed siRNA-PEI polyplexes. In contrast to our initial speculation, however, introduction of miRNA to a preformed siRNA-PEI polyplex revealed formation of a miRNA layer surrounding the polyplex through interactions with PEI. The resulting structure is a ternary siRNA-PEI-miRNA complex, where the experimentally determined ζ-potential was found to decrease as a function of miRNA added.
Asunto(s)
Portadores de Fármacos/química , MicroARNs/química , Simulación de Dinámica Molecular , Polietileneimina/química , ARN Interferente Pequeño/química , Conformación de Ácido Nucleico , Tamaño de la PartículaRESUMEN
Delivery of polynucleotides into patient cells is a promising strategy for treatment of genetic disorders. Gene therapy aims to either synthesize desired proteins (DNA delivery) or suppress expression of endogenous genes (siRNA delivery). Carriers constitute an important part of gene therapeutics due to limitations arising from the pharmacokinetics of polynucleotides. Non-viral carriers such as polymers and lipids protect polynucleotides from intra and extracellular threats and facilitate formation of cell-permeable nanoparticles through shielding and/or bridging multiple polynucleotide molecules. Formation of nanoparticulate systems with optimal features, their cellular uptake and intracellular trafficking are crucial steps for an effective gene therapy. Despite the great amount of experimental work pursued, critical features of the nanoparticles as well as their processing mechanisms are still under debate due to the lack of instrumentation at atomic resolution. Molecular modeling based computational approaches can shed light onto the atomic level details of gene delivery systems, thus provide valuable input that cannot be readily obtained with experimental techniques. Here, we review the molecular modeling research pursued on critical gene therapy steps, highlight the knowledge gaps in the field and providing future perspectives. Existing modeling studies revealed several important aspects of gene delivery, such as nanoparticle formation dynamics with various carriers, effect of carrier properties on complexation, carrier conformations in endosomal stages, and release of polynucleotides from carriers. Rate-limiting steps related to cellular events (i.e. internalization, endosomal escape, and nuclear uptake) are now beginning to be addressed by computational approaches. Limitations arising from current computational power and accuracy of modeling have been hindering the development of more realistic models. With the help of rapidly-growing computational power, the critical aspects of gene therapy are expected to be better investigated and direct comparison between more realistic molecular modeling and experiments may open the path for design of next generation gene therapeutics.